Method for the preparation of a composition of matter having antitumor and antifungal activity



United States Patent 1 3,117,916 METHOD FGR THE PREPARATION OF A COMPO-SITEON {1F MATTER HAVMG ANTITUMGR AND 2 J'IIFUNQAL ACTIVZTY Joseph FrankProkop, Mundelein, 111., assignor to Abbott Laboratories, North Chicago,111., a corporation of Illinois No Drawing. Filed May 22, 1952, Ser. No.196,618

5 Claims. (Cl. 1958(l) This invention is concerned with a novel compoundpossessing antiturnor and antifungal properties and to a process for itsproduction. In particular, the invention relates to a new composition ofmatter referred to herein as antitumor agent M-741, to a process for itsproduction by fermentation and to a method for its recovery,concentration and purification.

The principal object of the invention is to provide a new and usefulcomposition of matter which is active in suppressing the growth of avariety of tumors. Another object is to provide a process for thepreparation and recovery of M74l.

It has been found that by cultivating under controlled conditions and onsuitable culture media a heretofore undescribed microorganism namedStreptoverticillz'um sepratum, a novel composition of matter hereinidentified as antitumor agent M-741 is obtained. The micro organismemployed was isolated from a soil sainple collected near Shiremanstown,Pennsylvania. A culture of the living organism has been deposited and isavailable at the Culture Collection Unit of the Northern UtilizationResearch and Development Division, US. Department of Agriculture,Peoria, Illinois, under the code num ber NRRL 2974. The completetaxonomy of the new isolate follows.

TAXONOMY OF S TREPT OVERT I CILLI UM SEPTA TUAI With the exception ofdetermining cultural charac- Patented Jan. 14, 'lfihl 2 teristics andproteolytic activity in gelation which was done at 24 (3., all culturalcharacteristics in the standard media listed below were obtained byincubation at 2 C.

The color code references, such as 3 cc, are those from the ColorHarmony Manual, Third Edition, Jacobson, R.; Granville, W. C.; and Foss,C. E.; 1948; Container Corporation of America. The color names used arethose designated in the ISCCNBS Method of Designating Colors and aDictionary of Color Names, US. Department of Commerce, National Bureauof Standards, Circular 553, issued November 1, 1955. As an example, thecolor of the removable plastic color chip coded 3 co in the ColorHarmony Manual is named light grayish yellowish brown on page 52 of theNational Bureau of Standards Circular 553. In the following text ofcultural characteristics, color names from the National Bureau ofStandards Circular 553 are capitalized followed by the code of thematching color chip from The Color Harmony Manual in parentheses. Othercolor observations are not capitalized.

The morphological features of the organism hereinafter described placesit with the group of organisms hitherto known as Streptomyces withbiverticillate sporophores. However, in keeping with the proposal ofBaldacci, E., Giorn. Microbiol. 6, 10-27, 1958, that actinomycetes withverticillate sporophores be classified under a new genus Streptoverticilium, the organism is assigned to this genus. Moreover, a survey of theliterature failed to reveal species which satisfactorily agreed withcharacteristics of the organism hereinafter described. Accordingly, itis deemed to be a new species and is named Streptoverticillz'umseptatum. The specifi epithet refers to its characteristic septateaerial mycelium as will be described below.

Growth Characteristics of Streptoverticillium septatum [15 days agarplates, 28 0., except when noted] Medium Growth Aerial MyceliuinSporulation Substratal (Reverse) Soluble Notes-Physiologic Pigment andOther Complex media:

Nutrient agar. Good. Isolated 0010- None. 21 days None Dark yellow (2le) Dark yellow Occasional wisps or nies 4.0 mm. spread- (2 le) faint.slender corenua ooing over surface from served mna'oscoprflattenededges. cally. Glucose agar Excellent. Spreading Sparse. Yellowish None.21 Dark yellow (2 le) at do Abundant aerial wlsps 10.0 mm. over surgray(2 ob) at ed e, days. edge to moderate or long slender core- !ace fromconfluent light brownish gray olive brown (2 pi) Ima (aerial aggregrowthstreak. (3 to) center conflucenter confluent gates),

out growth. growth. e v Yeast-malt Excellent and rapid. Abundant,flutly, yel- Fair to Center, moderate yel- Very taint, AbundantIIIICIOGXLI- extract Spreading flatly lowish gray (2 ob). moderate.lowlsh brown (3 n); dark yellow date. agar. over surface from edge llghtolive (2 le).

edge of colonies. brown (2 ng). Soluble starch Good. N o well iso-Moderate. fiuft'y 15 Sparse Darlr grayish yellow Questlonable Hydrolysisstrongly yeast exlated colonies. days, abundant 21 (2 1e). to none.positive. tract, salts daysyellowish agar. y Defined media:

Slow. Low convex Very sparse, white. None through do None Rare wispsobserved colonies, flat edge, days. microscopically. slight radialwrinkl- 2.0 mm. zone of ming, slight spreading creased transparency oversurface of agar. of medium at edge of growth. Dextrose Initially slowbecom- Fluffy, moderate to Fair to Moderate olive (1% do Abundantmlcrodrops asparagine ing good. Isolated good. Pale yellow moderate.lg). of clear exudate 21 agar. colonies 4.0 mm. green (1 ha) 15 daysdays.

spreading over surto yellowish gray (2 face. ch) 21 days. v CalciumFair. Isolated colo- Sparse, white None through do No visible clearingmalate nies flat, 2.0 mm., 21 days. through 21 days. agar. entire edges.Inorganic Initially slow, becom- Abundant, fluffy 21 Fair Darlr grayishyellow do Hydrolysis strongly salts, ing good. days yellowish gray (21e). positive. soluble (2 ob). starch agar. Modified Abundant. IsolatedAbundant, flufiy or Abundant Grayish yellow (2 ge) do Growth extremelylun- Prldham colonies 8.0 mm. full cottony, light grayited in samemedium and Gottaerial. ish yellowish brown with CuSO at relteb basal (3cc). commended level. with dextrose (CuSO omitted).

PHYSIOLOGIC CHARACTERISTICS Synthetic nitrate agarno growth. Nutrientnitrate agarabundant growth. Test for nitrite negative at 2, 8 and 15days.

Tryptose agar with human bloodmarked hemolysis.

Plain gelatin-good growth, very weak liquefaction, dark brown solublepigment at top of plug.

Litmus milk-good growth, reaction unchanged, no coagulation, very slightpeptonization.

H productionweakly positive.

Starchhydrolyzed.

MICROMORPHOLOGY The fruiting structure is biverticillate in all media inwhich spores are produced although a rare chain of spores may be foundat the end of short branches arising singly from the main aerialfilaments. Primary verticils are short and sterile, spores beingproduced in secondary verticils. Uniformly the primary verticils ariseimmediately posterior to a septum in the aerial mycelium. On some media,primary verticils alone may be found typically branching from the aerialmycelium at the site of septation with no spores formed in the absenceof secondary verticils. The mycelium at the site of septation isslightly constricte This feature is best noted prior to and during theemergence of primary verticils.

Spore chains of the secondary verticils are short and devoid of books,loops, or spirals. The mature spores are predominantly bacil ary inmorphology with a low ercentage of oval forms being noted. The length towidth ratio averages 2:1 :as measured from electron micrographs. Thespore surface is smooth.

CARBOHYDRATE UTILIZATION Growth of S. septatzzm is severely restrictedin the basal medium of Pridham and Gottlieb, I. Bact. 56-: 107ll4, withthe addition of dextrose or glycerol. Following a metal tolerance study,the elimination of 0180 from the medium permitted good growth. Thefollowing table of carbohydrate utilization shows results obtained inthe basal medium modified by the elimination of CuSO Carbohydratesources shown as unavailable the modi tied medium also failed to supportgrowth in the complete medium.

The present invention embraces a process for growing Strcptoverticilliumseptazztm under controlled conditions which include a temperature of 24to 32 (1., submerged fermentation with suitable agitation and aerationusing a medium consisting of a carbon source such as glucose, glycerol,trans-esterified vegetable oils, or a combination of these; a source oforganic nitrogen such as soybean meal; a source of growth substances andminerals such as distillers solubles or corn steep liquor; mineral saltssuch as sodium chloride; an insoluble buffering agent to prevent theaccumulation of acid such as calcium carbonate; and a nonto.;c defoamingagent such as transesterified vegetable oils or soybean oil plusmethylpolysiloxane anti oam or a polypropylene glycol such as thatmarketed under the trade name Polyglycol P2000 by the Dow ChemicalCompany, Midland, Michigan. When the growth of the organism has produceda satisfactory amount of antibiotic substance as indicated by assays invivo against Sarcoma in the Swiss mouse, the culture is filtered and theantiturnor agent recovered from the filtrate. In practice, since thisassay requires a prolonged period of time, the culture is filtered at atime when the activity is presumed to be present in satisfactoryamounts, and the recovery procedures carried out while waiting forresults of the Sarcoma 180 assay. A process involving the use of acation exchange resin will remove the activity from the filtrate, andthe activity can be readily eluted from the resin by the use of amineral acid. Since during the elution a reasonable approximation of theamount of activity present can be obtained by measuring the opticaldensity of the eluate at between 275 and 280 mu, it is often best toprocess a small aliquot of a culture filt ate through the elution fromthe cation exchange resinand measure the optical density of the eluateand use this data to decide whether there is a satisfactory amount towarrant processing the entire filtrate. After elution from the cationexchange resin, the antibiotic can be further purilied by dialysis andby precipitation from the concentrated and acidified dialyzed solutionby a water miscible solout such as acetone, methanol or ethanol.

lnocuium suitable for use in shaken flasks can be obtained by using thegrowth from tryptone agar slants. This medium can also be used tomaintain, by transfer from slant to slant, suitable viable cultureswhich produce the antiturnor substance. However, in general practice,the maintenance of the Streptoverticillium septatum in soil or underlyophilization has proven a more dependable procedure. The growth onagar slants is used to inoculate shaken flasks which in turn may be usedto inoculate ferinentors of the size used for research purposes.

For production in a 23 liter fermentor, a satisf etory seed can beproduced by first transferring from an agar to a shaken flask andpermitting the flask to incubate on a rotary shaker for 2 days and usingthis shaken culture to inoculate suflicient shaken flasks to provide asatisfactory volume of inoculum for the 23 liter fermentor. This secondpassage of shaken culture is also continued for 2 days. However,satisfactory growth for inoculurn purposes can be obtained in 24 hours.In general, the production of the antit-umor agent in the 23 literfermentor reaches satisfactory yields in 3 to 4 days, and there is noadvantage in extending the fermentation beyond the 4 days. erobicconditions are maintained in the fermentors by forcing sterile airthrough a dispersing device in the bottom of the fermentor. The rate ofair forced into the culture medium varies somewhat with the size andshape of fermentation vessel. An aeration rate of V5 volume to onevolume of air per volume of culture per minute is satisfactory. Foamingof the culture medium during fermentation may be controlled withnon-toxic vegetable oils, such as transesterified vegetable oils, amethyiipolysiloxane antifoarn dissolved in a vegetabie oil such assoybean oil, or a polypropylene glycol such as Polyglycol P2000.Throughout the fermentation period, the culture medium is vigorouslyagitated by stirring devices which are part of the fermentation units.The degree of agitation is dependent upon the design of the varied sizedfermentation vessels, since it is well understood that pilot andcommercial sized fermentation tanks are designed for general usagerather than for a specific fermentation process. While particulars havebeen given for 23 liter fermentors, it should be recognized, as anyoneexperienced in the art will know, that the fermentation may be carriedout, with the usual modifications, in smaller fermentors or infermentors reaching up to the sizes used in commercial some productions.The organism Strepfoverzicillium sepmtum is able to produce the desiredantituinor agent in satisfactory amounts in a limited variety of culturemedia, over a temperature range of at least 2432 C.,

5 and it is apparently not necessary to maintain an exact aeration or aprecise amount of mechanical agitation.

The following examples illustrate the formation, recovery,concentration, and purification of antitumor agent M-741. These examplesare merely illustrative in nature and are not to be construed aslimiting.

Example 1 FERMENTATION IN SHAKEN FLASK Streptoverticillium septatum wasseeded from soil stock to an agar slant of the following composition andincubated for about five days at 28 C.

Tryptone "grams" 3 Beef extract do 3 Cerelose (glucose) do 1 Yeastextract do 1 Agar do 15 1-1 liters 1.0

After incubation, the resulting growth was scraped from the agar slantand introduced into 150 m1. of medium of the following composition in a500 m1. Erlenmeyer flask.

Soybean meal grams 15 Cerelose (glucose) -do 15 NaCl do CaCO do 1 H Oliters 1 This flask was incubated for 48 hours at 28 C., on a rotaryshaker describing a 2% inch eccentric at about 240 rpm. Following this,another flask containing sterile medium of the same composition wasseeded With about 4% v./v. of the resultant growth from the first flask.This flask was also incubated for 48 hours at 28 C. with shaking.

For production, a 500 ml. Erlenmeyer flask containing 150 ml. ofsterilized medium of the following composition was seeded with 4% v./v.of material from the second 48 hour flask. Incubation and agitation forthis flask was as before with the exception that the incubation periodwas for approximately 72 hours.

Soybean meal grams 1O erelose (glucose) do NaCl do 5 CaCO do 1 H Oliters 1 After incubation, the resultant material was harvested and thefiltrate tested for activity against Sarcoma 180 in mice with thefollowing results:

Dilution Wt. T/C, Test No. for dosage change, percent gram 1 Difierencein average weight gain or loss in controls and that of test animals.

aver-age tumor Weight of test animals as a percent of average tumorweight of controls.

Example 2 Glucose monohydrate Soya-flulf flour (finely ground defattedsoybean meal) 15 Sodium chloride 5 Calcium carbonate 1 The flask and itscontents are sterilized by autoclaving for a period of to minutes at atemperature of 121 C. After cooling, the flask is inoculated .with asection from the surface of a tryptone agar slant on whichStreptoverticillium septatum has been growing for at least 3 days. Theinoculated flask is agitated at 28 C. on a rotary shaker having a strokeof 2% inches and operating at about 230 rpm. for a period of 48 hours. Asecond passage of the seed culture is prepared by using the aboveculture to inoculate additional flasks prepared and sterilized as above.Each flask is inoculated with about 3 ml. of the 48-hour culture. Theseed flasks are incubated and agitated as just described for 48 hours.

In a fermentation tank of 23-liter capacity is placed 12 liters of afermentation medium having the following composition:

Grams per liter Glucose monohydrate 25 Soybean meal 20 Calcium carbonate1 Sodium chloride 5 To the above is added 0.1% by volume of PolyglycolP-2000 as an antifoam agent. The fer-mentor zmd its contents aresterilized by :autoclaving for 75 minutes at 121 C. After cooling, thefermentor is inoculated aseptically with the contents of three of theabove described flasks of second passage seed culture. The culture isgrown in the fermentor at 28 C. for 3 days during which time the brothis stirred mechanically and sterile air is passed into the bottom of thetank at the rate of about 0.8 volume of air per volume of broth perminute. The maximum antiturnor activity is reached after about 3 days.The presence of the antitumor agent is demonstrated when a 1:128dilution of a sample of filtered beer markedly inhibits the growth oftransplanted Sarcoma 180 tumor in the Swiss mouse, when injected dailyat a rate of 1 ml. per day over a six day period. Under these conditionsthe growth of the tumor in the treated mice averages, in three separatetests, 27%, 51%, and 20% of the growth of tumors in untreated controls.

RECOVERY on ANTITUMOR AGENT M-741 FROM 23-LITER FERMENTOR BEER About 120liters of beer produced as in Example 2 are filtered with suction toyield about liters of filtrate. The filtered beer is passed over acolumn 3 in diameter and 48" in height of a cation exchange resincomposed of a cross-linked copolymer of methacrylic acid and divinylbenzene, containing between 4 and 10 mol percent of divinyl benzene,marketed under the tradename iRC-SO, in the sodium form. Those familiarwith the art will recognize that the process is not limited to thisparticular resin, but that other cation exchange resins of similar ordiverse nature can also be used. After the beer has gone through thecolumn, the column is washed with 58 liters of deionize dwater anddeveloped with 0.5 N HCl. Here again, those familiar with the art willrecognize that developm nt is not necessarily limited to this normalityof HCl or to this particular mineral acid. During the development, theeffluent is sampled regularly and the optical density of these samplesat 275 mu is measured. When the optical density has reached 1.0,fractions are collected and pooled while the optical density rises to amaximum greater than 2.0 and drops back to 1.25. Fractions can becollected after this point, while there is a gradual drop in thedensity, but there is a sharp rise in impurities, and in practice it isbetter to accept a small loss in antitumor activity and avoid a largeincrease in impurities. The pool of the high optical density fractionshas a volume of between 8 and 9 liters. This is concentrated, forconvenience, on a vertical tube evaporator under vacuum at a temperatureof less than 32 C. to a volume of 2 liters. The optical density of thisconcentrate at 275 mu is 11.70.

This material is dialyzed against running deionized water. Afterdialysis, the volume has expanded to about 3500 ml. and the solution nowcontains a precipitate.

This precipitate is separated by centrifugation and is furth 1 purifiedby extraction with two 25 ml. portions of 0.02 N l-lCl. The insolublematerial is removed by centrifugation, and to the clear supernatant isadded about 7 volumes of acetone. A white precipitate forms and thesuspension is allowed to stand at about 4 C., overnight. The precipitateis separated by filtration and washed on the paper with first about 50ml. of a solution composed of 7 parts acetone and 1 part deionized waterand finally with 50 ml. of dry acetone. The precipitate is then removedfrom the paper and dried overnight in a vacuum dessicator overconcentrated H 30 The weight of this precipitate is 564.7 mg. Thisprecipitate, Precipitate l, inhibits the development of transplantedSarcoma 180 in the Swiss mouse to 22% of that in untreated controls wheninjected over a 6 day period at a level of 0.062 mgJkg/day.

The original 3500 ml. of dialyzed eluate after the removal of the abovedescribed precipitate is concentrated a vertical evaporator to 250 ml.Late in the concentration, another precipitate appears which is alsoremoved by centrifug tion. This precipitate is extracted once with a 25ml. portion of 0.02 N HCl. Only a trace of solid material is left. Thisis removed by centrifugation and to the supernatant is added 7 volumesof acetone. A white precipitate appears. This is also allowed to standat about 4 C overnight, is separated by filtration, is washed with about50 ml. of a solution composed of 7 parts acetone and 1 part deionizedwater followed by 50 ml. of dry acetone. It is removed from the paperand dried overnight '11 a vacuum dessicator over concentrated H SO4. Theyield of amorphous slightly otf-white powder is 122.1 mg. Thisprecipitate, Precipitate ll, inhibits the development of transplantedSarcoma 180 in the Swiss mouse when injected over a 6 day period at 0.25mg/kg/day to 18% of that in untreated controls.

The 250 ml. or supernatant-from which the above described precipitatehas been obtained is further purified by adding ml. of 1.0 N HCl, sothat the solution is now a 0.02 N l-iCl solution. Seven volumes ofacetone are now added. A White precipitate appears. The suspension isallowec to stand at about 4 C. for about 12 hours, after which theprecipitate is removed by filtration, Washed with 250 ml. of a 7 partsacetone and 1 part deionized water solution, and finally washed withabout 250 ml. acetone. The precipitate is removed from the paper anddried overnight in a vacuum dessicator over concentrated H 59 The yieldof amorphous sli htly oil-white powder is 3.2851 grams. Thisprecipitate, Precipitate Ill, inhibits the development of transplantedSarcoma 180 in the Swiss mouse when injected over a 6 day period at0.084 rug/kg] day to 20% of that in untreated controls.

All preparations show similar ultra-violet curves with weak maxima at278 mu and an extinction coefi'icient of llm. 17

BIOLOGICAL ACTIVITY OF M741 The following microorganisms are notinhibited in their growth on agar containing 100 micrograms/ m1. ofM741.

Agrobacterium tumcfaciens Aspergillus niger Bacillus subrz'lis Candidaalbicans Erwz'nia amylovora Escherichia coli F usarium oxysporumKlebsiclla pneumoniac M'ycobacrerium smegmatis Neisserz'a catarrhulisProteus vulgaris Pseudomonas tabacz' Salmonella typlzosa Staphylococcusaureus Streptococcus faecalis Xantlzomonas plmseoli Aantlzomonasvcsicatoria In a tube dilution assay, the following microorganisms arenot inhibited in their growth by the presence of 1000 micrograms/ml. ofM-741:

Escherichia coli Proteus mirabilis Proteus vulgaris Pscudomorzasacruginosa Salmonella typhimurium Staphylococcus aurcus, Smith Thefollowing microorganisms are not inhibited by 200 meg/ml. of M-741:

Klcbsiella pneumonz'ae Salmonella typhosa Staphylococcus aurcus, WiseStreptococcus faecalis The following fungi are inhibited by the presenceof ltd-741 in agar at the concentrations indicated:

Fungus tested: lillll, fii llilhilili Altcrnaria species 1,000Aspergillus versz'color 1,000 Chaetomium globosum 500 Fusarium oxysporum1,000 Myi'orizccium verrucaria 500 Penicillin citrinum 5,000

ANTITUMOR PROPERTIES OF M-74l M741 shows activity in varying degreeagainst a wide variety of transplantable tumors in the mouse and rat.These are listed below.

m Dose level at which 31-741 1 llmgris active (mg./kg./day) Sarcoma 0.2Sarcoma 180 (ascites form) 0.1 Mammary adenocarcinoma EOTH 0.1 Miyonoadenocarcinoma 0.1 Ridgeway osteogenic sarcoma 0.1 Carcinoma 1025 0.2Bashford carcinoma 63 0.1

Ehrlich carcinoma 0.1 Ehrlich carcinoma (ascites form) 0.1 Lewis lungcarcinoma 0.2 Gliorna 26 0.2 Harding-Fares melanoma 0.2 Walker carcinoma255 0.2 Murphy-Sturm lymphosarcoma 0.2 Flexner-Jobling carcinoma 0.2Novikoi'l rat hepatoma 0.2 Wagner osteogenic sarcoma 0.025 L4946 0.1Iglesias sarcoma 0.2

What is claimed is:

1. A process for the production of a composition of matter havingantitumor and antifungal activity which com rises cultivating theorganism Str eptoverricillium scptatum under submerged aerobicconditions in a culture medium containing assimilable sources ofcarbohydrates, organic nitrogen and inorganic salts until substantialantiturnor activity is produced by said organism and recovering theresultant product from said culture medium.

2. A process as claimed in claim 1 in which the organism employed is Streproverzicz'llium septatum NRRL 2974.

3. A process as claimed in claim 2 in which the culture medium ismaintained at a temperature of from 24- 32 C. for a period of from 2 to4 days.

4. A process as claimed in claim 2 which includes the steps ofclarifying the culture medium, adsorbing the active component therefromwith a solid adsorbent and eluting the adsorbate.

5. The product produced by the process of claim 1.

No references cited.

1. A PROCESS FOR THE PRODUCTION OF A COMPOSITION OF MATTER HAVINGANTITUMOR AND ANTIFUNGAL ACTIVITY WHICH COMPRISES CULTIVATING THEORGANISM STREPTOVERTICILLIUM SEPTATUM UNDER SUBMERGED AEROBIC CONDITIONSIN A CULTURE MEDIUM CONTAINING ASSIMILABLE SOURCES OF CARBOHYDRATES,ORGANIC NITROGEN AND INORGANIC SALTS UNTIL SUBSTANTIAL ANTITUMORACTIVITY IS PRODUCED BY SAID ORGANISM AND RECOVERING THE RESULTANTPRODUCT FROM SAID CULTURE MEDIUM.